Members of the trypsin/chymotrypsin-like (S1) serine protease family play pivotal roles in a multitude of diverse physiological processes, including digestive processes and regulatory amplification cascades through the proteolytic activation of inactive zymogen precursors. In many instances protease substrates within these cascades are themselves the inactive form, or zymogen, of a xe2x80x9cdownstreamxe2x80x9d serine protease. Well-known examples of serine protease-mediated regulation include blood coagulation, (Davie, et al. (1991). Biochemistry 30:10363-70), kinin formation (Proud and Kaplan (1988). Ann Rev Immunol 6: 49-83) and the complement system (Reid and Porter (1981). Ann Rev Biochemistry 50:433-464). Although these proteolytic pathways have been known for sometime, it is likely that the discovery of novel serine protease genes and their products will enhance our understanding of regulation within these existing cascades, and lead to the elucidation of entirely novel protease networks.
Proteases are used in non-natural environments for various commercial purposes including laundry detergents, food processing, fabric processing and skin care products. In laundry detergents, the protease is employed to break down organic, poorly soluble compounds to more soluble forms that can be more easily dissolved in detergent and water. In this capacity the protease acts as a xe2x80x9cstain remover.xe2x80x9d Examples of food processing include tenderizing meats and producing cheese. Proteases are used in fabric processing, for example, to treat wool in order prevent fabric shrinkage. Proteases may be included in skin care products to remove scales on the skin surface that build up due to an imbalance in the rate of desquamation. Common proteases used in some of these applications are derived from prokaryotic or eukaryotic cells that are easily grown for industrial manufacture of their enzymes, for example a common species used is Bacillis as described in U.S. Pat. No. 5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serine proteases isolated from a fungus, Tritirachium album, for use in laundry detergent compositions. Unfortunately use of some proteases is limited by their potential to cause allergic reactions in sensitive individuals or by reduced efficiency when used in a non-natural environment. It is anticipated that protease proteins derived from non-human sources would be more likely to induce an immune response in a sensitive individual. Because of these limitations, there is a need for alternative proteases that are less immunogenic to sensitive individuals and/or provides efficient proteolytic activity in a non-natural environment. The advent of recombinant technology allows expression of any species"" proteins in a host suitable for industrial manufacture.
Herein we describe a novel serine protease isolated from small intestine termed D-G. The deduced amino acid sequence encodes a polypeptide of 435 amino acids. Interestingly, the sequence contains a hydrophobic stretch of amino acids which is a putative transmembrane near the NH2-terminus. Thus, this serine protease is thought to be synthesized as a type II integral membrane protein. Alignment with other well characterized serine proteases clearly indicates that it is a member of the S1 serine protease family with the catalytic triad residing within the C-terminal half of the molecule. The protease D-G deduced amino acid sequence is most similar to the cloned serine proteases TMPRSS2 (Paoloni-Giacobino et al. (1997). Genomics 44:309-320) and hepsin (Leytus et al. (1988). Biochemistry 27:1067-74), which are also type II integral membrane proteases. We have found that the protease D-G mRNA is widely expressed in several tissues throughout the body including epidermis, fibroblasts, keratinocytes, colon, small intestine, stomach, lung, kidney, bone marrow, lymph node, thymus, ovary, prostate, uterus and spinal cord. Altered expression or regulation of this enzyme may be responsible for any one of a number of pathological conditions in these tissues. Furthermore, an up-regulation whereby under normal physiological conditions protease D-G mRNA is not expressed, and therefore undetected, but in the pathogenic condition it is markedly elevated could potentially result in initiating or exacerbation of certain diseased states. We expressed a soluble form of this novel human protease by inserting the portion of the protease D-G cDNA, encoding the catalytic domain, in a zymogen activation construct designed to permit the generic activation of heterologous serine protease catalytic domains. The result is an active preparation of protease D-G that has an activity against a subset of amidolytic substrates. Isolation of purified, enzymatically active protease D-G allows the protein to be used directly, for example to discover chemical modulators of the enzyme or as an additive in commercial products. Because protease D-G is derived from a human host, it is less likely to induce an allergic reaction in sensitive individuals, and therefore protease D-G may also be useful for formulation of compositions for laundry detergents and skin care products.
A DNA molecule encoding protease D-G has been cloned and characterized and it represents a novel serine protease. Using a recombinant expression system functional DNA molecules encoding the protease have been isolated. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules. The recombinant protein is useful to identify modulators of functional protease D-G. Modulators identified in the assays disclosed herein may be useful as therapeutic agents for cancer, skin disorders, neuropathic pain, inflammatory, or coagulation diathesis/thrombosis.